The present invention relates to the field of gene expression in plants and in particular concerns gene silencing, a phenomenon frequently observed after integration of transgenes into plant genomes. Comparison of transcriptional gene expression between an Arabidopsis line carrying a silent transgene present in multiple copies and its mutant derivative mom1 impaired in silencing of the transgene revealed two cDNA clones which are expressed in the mutant plants, but not in the parental and not in wild type plants. Both clones are derived from the same family of transcripts which we refer to as TSI (Transcriptionally Silent Information). The disclosed genomic templates encoding TSI are repetitive elements with mainly pericentromeric location and conserved organizaton among various ecotypes. They are also referred to as TSI. Transcriptional silencing of the genomic TSI templates is specifically released in the mutant. Silencing of said templates is further released in other genotypes known to affect transcriptional gene silencing. Thus, transcription of TSI can be used as a marker to identify a defective silencing pathway in a plant.
Correct balance between activation and silencing of its genetic information is essential for any living cell. A tight control of gene expression is necessary for adaptation to environmental factors, regulation of physiological requirements, and development of differentiated, specialized cell types within a multicellular organism. For example differentiation processes involve mitotically heritable changes of gene expression, wherein the acquired states of gene activity gain a certain stability. This stability can be achieved by the strict control of gene activators, by regulation of transcript stability, or by regulating the transcriptional availability of genetic information itself as by stable silencing of selected genetic loci. Silencing has been frequently observed in connection with repression of transgene expression in various experimental systems.
In plants, silencing of transgenic loci limits the reliability of transgenic approaches to improve quality traits. It has been noticed that complex inserts containing rearranged multiple copies of a transgene are particularly prone for gene silencing. Two different mechanisms leading to loss of transgene expression are observed. The first prevents transcription (transcriptional gene silencing or TGS), and the second targets selected transcripts for rapid degradation (posttranscriptional gene silencing or PTGS). Triggers of both processes seem to be similar, since the onset of both types of silencing correlates with redundancy of genetic information, i.e. DNA repeats in case of TGS and RNA overproduction for PTGS. TGS is meiotically heritable and correlates with DNA template modification manifested by hypermethylation of promoters of silenced genes or with local changes of chromatin structure. In contrast, PTGS is not meiotically transmitted and needs to be reestablished in each sexual generation. PTGS does not require modification of a DNA template, however, increased levels of DNA methylation within the protein-coding region of silenced genes have been observed.
The majority of silencing studies in plant systems deal with silencing of transgenes. There are only a few examples of gene silencing without involvement of transgenic loci. The criteria for TGS susceptibility of genetic information is very poorly understood, and the natural targets of transcriptional silencing in a normal, wild type plant are yet to be discovered. It has been postulated that TGS is a defense system against invasive DNA such as transposable elements but experimental evidence for this hypothesis is lacking.